Pharmaceutical Composition Containing Expanded Adult Stem Cells and Methods of Using Same for Treatment

ABSTRACT

A method for the expansion of adult stem cells from blood, particularly but not only peripheral blood, involves removing adult stem cells from blood of a mammal, immediately expanding the stem cells via in-vitro treatment with MCSF (Macrophage Colony Stimulating Factor) at a concentration of about 8-15 nM, and purifying the expanded stem cells. Compositions and methods of using the expanded adult stem cells are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of allowed U.S. patent application Ser.No. 12/408,145, which was a Continuation-in-Part of InternationalApplication No. PCT/EP2007/059531, filed Sep. 11, 2007, which waspublished in the English language on Mar. 27, 2008 under InternationalPublication No. WO 2008/034740, and the disclosure of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention concerns a method for expanding stem cells fromblood, particularly but not only peripheral blood, of adult mammals, andthe relative application in the medical field, in particular in theveterinary field, for the treatment of lesions, chronic and/or acuteinflammatory pathologies, and neurological and neurodegenerativepathologies. Here, and hereafter in the description, and as is known inliterature, the terms “expanding” and “expansion” refer to the processof increasing the number of cells either by cell division or, as in thespecific case described and claimed, by “de-differentiation”, that is,the process by which the cells present in the blood are transformed intostem cells following suitable in-vitro treatment, as will be seenhereafter.

In recent years the use of stem cells in therapy has had great approvaldue to the successes obtained in treating various pathologies which werepreviously thought incurable. However, processes known until now forobtaining stem cells have been laborious and expensive.

Pluripotent stem cells (PSC) are a source available not only forresearch but also for the creation of drugs and for transplants (A. J.Wagers et al., “Cell fate determination from stem cells”; Gene Therapy;9; 606-612 (2002); L. G. Griffith et al., “Tissue Engineering—CurrentChallenges and Expanding Opportunities”; Science; 295; 1009 (2002)).

There are two ample categories of stem cells: embryonic and adult. Theformer are derived from embryos, and more exactly from 8-dayblastocysts, whereas adult stem cells may be obtained mainly from bonemarrow, adipose or muscular tissue, or from peripheral blood.

The definition of stem cell is constantly evolving and, at the moment,there is no general consensus or standard method to isolate or identifythem. For all these cells, embryonic (ES) and adult, both hematopoietic(HSC) and mesenchymal (MSC) (M. Kuwana et al., “Human circulating CD14⁺monocytes as a source of progenitors that exhibit mesenchymal celldifferentiation”; J. Leuk Biol; 74; 833-845 (2003)), different geneticmarkers have been identified, of which some are common to many celltypes. (See, for example M. Condomines et al., “Functional Regulatory TCells Are Collected in Stem Cell Autographs by Mobilization withHigh-Dose Cyclophosphamide and Granulocyte Colony-Stimulating Factor”;J. Immunology; 176: 6631-6639 (2006); W. J. Kang et al., “TissueDistribution of ¹⁸F-FDG-Labeled Peripheral Hematopoietic Stem CellsAfter Intracoronary Administration in Patients with MyocardialInfarction”; J. Nucl Med.; 47:1295-1301 (2006); Y. Zhao et al., “A humanperipheral blood monocyte-derived subset acts as pluripotent stemcells”; PNAS; vol. 100, No. 5; 2426-2431 (2003); and M. Rabinovitch etal., “Cell Shape Changes Induced by Cationic Anesthetics”; J.Experimental Medicine; 143; 290-304 (1976)).

In particular, Zhao Y. et al. (“A human peripheral bloodmonocyte-derived subset acts as pluripotent stem cells”; PNAS; vol. 100,No. 5; 2426-2431 (2003) and WO 2004/043990) discloses a method forpreparing monocyte-derived stem cells which includes the steps ofisolating a peripheral-blood monocyte, contacting it with a mitogeniccomponent, and subsequently culturing the peripheral-blood monocyteunder conditions suitable for the propagation of the cells.

This method, which requires a first step of isolating the monocyte and asubsequent expansion step in a culturing media, requires very longtimes, on the order of 15-20 days, to obtain a significant number ofstem cells. By this method, it is not possible to obtain stem cells ofthe totipotent type, that is, cells that are non-specialized andsuitable to be directly inoculated into the patient after a very shorttime from the first drawing.

Numerous scientific works describe stem cells' ability to regeneratedifferent types of lesions by regenerating tissues that are mechanicallydamaged or are damaged by various pathologies, thus eliminating at theroot the causes that generated the pathology and not simply acting onthe effects thereof.

At the moment, research is more oriented toward the use of stem cellsisolated from embryonic tissue, fetuses, or the umbilical cord, but thiswork is raising various legal and ethical questions. Above all, as oftoday the use of these cells brings various contraindications, such asrisk of infection, risk of rejection if transplanted and, in horses, therisk of onset of teratomas.

To obviate these problems, it has therefore been contemplated to use inthe “in vivo” therapy autologous stem cells isolated preferably frombone marrow, adipose tissue or peripheral blood. These methods, startingfrom adult stem cells, provide a step of differentiation “in-vitro” (or“ex vivo”) of the stem cells in the cell line desired by means ofspecific differentiation induction factors, and a subsequent step of “invivo” transplantation of the differentiated cell line obtained. In thesemethods, the limit is due to the fact that observable rejectionphenomena occur because the differentiated cells re-introduced into thepatient are not recognized as self-cells, but lose the self-recognitionfactors during the differentiation step induced in-vitro.

In humans, taking stem cells from peripheral blood entails purifyingthem through a process called “aphaeresis” or “leucophaeresis”. Inpractice, the cells are extracted from the blood, collected, and theninoculated into patients immediately after chemo- or radio therapy. Inaphaeresis, which lasts from 6 to 8 hours, the blood is taken from thevein of an arm or a vein in the neck or the chest, and made to passthrough a machine which removes the stem cells. The blood, thuspurified, returns to the patient, while the cells collected arepreserved by means of refrigeration in liquid nitrogen (M. Condomines etal., “Functional Regulatory T Cells Are Collected in Stem CellAutographs by Mobilization with High-Dose Cyclophosphamide andGranulocyte Colony-Stimulating Factor”; J. Immunology; 176: 6631-6639(2006); W. J. Kang et al., “Tissue Distribution of ¹⁸F-FDG-LabeledPeripheral Hematopoietic Stem Cells After Intracoronary Administrationin Patients with Myocardial Infarction”; J. Nucl Med.; 47:1295-1301(2006)). This technique is not only painful, but also extremelystressful for the patient. Furthermore, it is impracticable for animalsof either small or large size; above all, the technique does not providea real discrimination and/or purification of the stem cells circulating.

At present, in veterinary science, stem cells are used successfullymainly in the reconstruction of tendons and ligaments with lesions. Themain techniques for purification include:

-   -   use of growth factors or platelet derivatives (TGF-B, VEGF), but        the economic costs of extracting these are prohibitive (M. Hou        et al., “Transplantation of mesenchymal stem cells from human        bone marrow improves damaged heart function in rats”;        International Journal of Cardiology; 115; 220-228 (2006));    -   isolation of stem cells taken from bone marrow. This technique        provides for purification and then use for therapy of only 15%        of the cells contained in the material extracted;    -   isolation of stem cells taken from adipose tissue. This        technique, which requires the prior surgical removal of        considerable quantities of tissue from the donor animal, does        not allow for intravenous administration;    -   IGF-1 (insulin-like growth factor 1), known as Tendotrophin (J.        Fiedler et al., “IGF-I and IGF-II stimulate directed cell        migration of bone-marrow-derived human mesenchymal progenitor        cells”; Biochemical and Biophysical Research Communications;        345; 1177-1183 (2006));    -   UBM (urinary bladder matrix), a derivative from the pig        containing cytokines (but not nucleate cells), which induces the        cicatrization of the wound but not the regeneration of the zone        with lesions (Y. S. Zhang et al., “Preliminary research on        preparation of porcine bladder acellular matrix graft for tissue        engineering applications”; Zhonghua Yi Xue Za Zhi; 85 (38);        2724-2727 (2005)).

In the light of all the above, it is obvious that methods are needed forthe expansion and purification of adult stem cells from easilyaccessible sources which must also provide for obtaining stem cellssuitable for use as medication in the medical-veterinary field. Onceadministered in the mammal, such cells do not give rise to phenomena ofrejection and are easy to preserve.

There is also an obvious need for obtaining stem cells of thepluripotent and totipotent type, that is, non-specialized cells, whichcan be inoculated directly into the patient with much shorter productiontimes than those provided at present.

BRIEF SUMMARY OF THE INVENTION

The authors of the present invention have now perfected a method for theexpansion and purification in-vitro of stem cells from peripheral blood.The method provides for obtaining stem cells which do not givecollateral effects, such as phenomena of rejection, infection, orteratomas. Preferably, once administered in the adult mammal, thesecells are able to be differentiated “in vivo” and to behave aspluripotent stem cells.

Applicants have seen that the cells thus expanded, once injected locallyor intravenously, acquire “in vivo” (and not “in-vitro”, as in knownmethods in the state of the art by means of suitable growth factorsand/or chemical stimuli (R. Gulati et al., “Diverse Origin and Functionof Cells with Endothelial Phenotype Obtained from Adult Human Blood”;Circ. Res.; 93; 1023-1025 (2003); M. Korbling et al., “Hepatocytes andEpithelial Cells of Donor Origin in Recipients or Peripheral-Blood StemCells”; New England Journal of Medicine; 346: 738-746 (2002); and T.Okazaki et al., “Macrophage Colony-Stimulating Factor Induces VascularEndothelial Growth Factor Production in Skeletal Muscle and PromotesTumor Angiogenesis”; J. Immunology; 174: 7531-7538 (2005)) all themorphological and chemical characteristics of macrophagic, lymphocytic,epithelial, endothelial, neuronal and hepatocyte cells, according to theneeds and pathologies of the animals treated. The method is even lessinvasive than other methods used until now to collect stem cells,painless (if compared with aphaeresis), economical, and technically themost suitable to be used in all animal species (small and large).

Finally, the possibility of obtaining these cells easily, and then beingable to preserve them for a long time refrigerated in liquid nitrogen,makes the cells obtained by the method according to the inventionsuitable for autologous transplants, for the treatment of many human andveterinary pathologies (including lesions of various types, metabolicillnesses, acute and chronic neurological and inflammatory pathologies)and for the improvement of competitive performances of some animals,such as horses.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiments which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown.

In the drawings:

FIG. 1 is photograph of a lesion of 20 cm diameter between themetatarsus and the first phalange with clostridia and destruction of theextensor tendon in a mare;

FIG. 2 is a photograph of the same mare of FIG. 1 three months after thelocal application to the lesion of the stem cells according to theinvention;

FIG. 3 is a photograph of the mare after six months of treatment of thelesion;

FIG. 4 is a ultrasound scan of a horse with a lesion of 80% of thesurface flexor tendon;

FIG. 5 is a ultrasound scan of the horse about three and a half monthsafter the local treatment with stem cells according to the invention;

FIG. 6 is a further ultrasound scan of the horse about three and a halfmonths after the local treatment with stem cells;

FIG. 7 is a ultrasound scan of the horse after about four months of thelocal treatment, depicting nearly complete regeneration of the tendonand the lack of scar tissue;

FIG. 8 is a ultrasound scan of a mare with a smaller tendon lesion ofless than 1 cm in diameter;

FIG. 9 is a ultrasound scan of the same mare in after a month of thelocal treatment by a method of the invention;

FIG. 10 is a photograph of a 17-year-old horse with general weakness,operated for colic, before treatment with the stem cells according tothe invention;

FIG. 11 is a photograph of a performance of the 17-year-old horse oneyear after intravenous treatment with the stem cells according to theinvention;

FIG. 12 is a graph of the number of cells obtained using differentquantities of MCSF in one embodiment of the present invention; and

FIG. 13 is a graph of the number of cells obtained from peripheral bloodv. time for cells pretreated with and not treated with MCSF.

DETAILED DESCRIPTION OF THE INVENTION

The present invention therefore specifically concerns a method forexpanding adult stem cells from blood, particularly but not limited toperipheral blood, comprising the following steps.

The first step in the method of the invention involves removing adultstem cells from the blood (preferably, but not limited to peripheralblood) of an adult mammal. Subsequently, the method involves expandingthe stem cells of blood immediately after they have been removed fromthe adult mammal via an in-vitro treatment with MCSF (Macrophage ColonyStimulating Factor) at a concentration of about 8-15 nM, preferablyabout 10 nM. The duration of the expansion step may vary according tothe conditions in which the in-vitro treatment is carried out.Applicants have determined experimentally that a duration of in-vitrotreatment with MCSF of about 24 to 96 hours, advantageously about 48 to72 hours, leads to a stabilization of the expansion with identificationof the simultaneous presence of the markers CD 90, CD34 and mixed CD90/CD 34. Accordingly, this duration is considered optimum, but otherdurations would also be within the scope of the invention.

The term “immediately” is intended to mean the shortest possible timebetween the blood cells being removed from the mammal and the beginningof the in-vitro treatment. In any case, the time should preferably benot more than about 10 minutes and advantageously not more than about 5minutes to avoid blood coagulation and to obtain the desired number ofstem cells number in the shortest amount of time between the removal ofthe blood sample and the final treatment.

Following the expansion, a further step in the method of the inventioninvolves purification of the expanded stem cells, preferably by means offractioning on a Ficoll gradient. This purification step isfundamentally intended to destroy the red corpuscles.

Therefore, contrary to the state of the art, the presently claimedinvention provides a step of expansion of the stem cells by contactingthem with MCSF and a possible anti-coagulant product, for example in asuitable test tube. This expansion step is performed immediately afterthe stem cells of blood have been removed from the patient, withoutisolating specific parts of them and without using any culturing media.A preferred embodiment of the present invention concerns the expansionmethod for stem cells from peripheral blood of adult mammals.

In a preferred embodiment, following the purification step, the purifiedcells are further expanded via in-vitro treatment with MCSF at aconcentration of about 35-55 nM, preferably about 50 nM, more preferablyabout 45 nM. This duration of this step may also vary from about 24 toabout 96 hours, preferably about 48 to 72 hours. In one preferredembodiment, at least one of the expanding steps is performed for about25 to 48 hours, preferably about 45 to 48 hours.

It has been observed that when MCSF is used at a concentration greaterthan about 55 nM (i.e., about 70 nM), after 24 hours the cells alreadydid not maintain the phenotype of pluripotent stem cells (see FIG. 12).Specifically, the graph in FIG. 12 depicts the number of cells obtainedusing different concentrations of MCSF: 15 nM (broken line withcrosses), 25 nM (broken line with triangles, apex down), 35 nM (brokenline with triangles, apex up), 50 nM (broken line with black circles),60 nM (broken line with white squares), and 70 nM (broken line withwhite circles). In each case, the results represent the average numberof cells counted from three samples of peripheral blood.

In particular, the first step of prior expansion in suspension with MCSFimmediately after the blood has been removed provides for an increase inthe percentage of stem cells (see FIG. 13). Specifically, the graph inFIG. 13 depicts the number of cells obtained from peripheral blood afterthe first expansion step according to the method of the invention. Thebroken line with dots represents cells that were pre-treated with MCSFwhereas the broken line with crosses represents cells that were nottreated with MCSF. In each case, the results represent the average ofcells counted from three samples of peripheral blood. The subsequentexpansion step in one embodiment of the invention provides for obtainingpluripotent stem cells which are differentiated directly in vivo,without causing phenomena of rejection or infection.

The invention also relates to a method of utilizing the expanded adultstem cells obtained according to the expansion method described abovefor the preparation of a medication for treating lesions of mammals. Thetreatable lesions include, without limitation, cutaneous lesions,lesions to tendons, lesions to ligaments, bone lesions, lesions to themucous membranes, and fractures.

The present invention also relates to a method of utilizing the expandedadult stem cells obtained according to the method described above forthe preparation of a medication for treating neurological orneurodegenerative pathologies in mammals, including, without limitation,Cushing's disease, head shaking, Wobbler's syndrome, breathingdifficulties, paresis of the limbs; acute or chronic inflammatorypathologies such as laminitis, periostitis, gastritis, arthrosis,inflammations caused by viral, bacterial, parasite, and/or mycoticagents; and dilatation-torsion of the stomach.

A method is also provided for utilizing the expanded adult stem cellsobtained by the method according to the invention for treatinginfertility in mares, precocity in colts and for improving competitiveperformance or activity in mammals.

The present invention also relates to a pharmaceutical compositioncomprising an active principle comprising the expanded adult stem cellsobtained according to the expansion method described above and at leastone pharmacologically acceptable adjuvant and/or excipient. Suchadjuvants and excipients are well known to those skilled in the art andneed not be described.

In a first particularly preferred composition, formulated forintravenous injection, the adult stem cells are contained at aconcentration of about 90-250×10³ cells/ml in the composition,preferably about 150×10³ cells/ml. A second preferred compositionaccording to the invention may be formulated for local application, aswell as for topical application in the case of external wounds. Such acomposition preferably comprises the adult stem cells obtained accordingto the expansion method defined above at a concentration of about4-40×10⁶ cells/ml in the composition.

The pharmaceutical compositions mentioned above may also comprise anantibiotic as an active principle at a concentration of about 5-15 nM,preferably 10 nM. Preferably, the antibiotic is gentamicin or amikacin.

The invention also relates to a method of using the secondpharmaceutical composition as described above (i.e., suitable for localapplication, with or without antibiotic) as a medication for treatinglesions in mammals, such as, but not limited to cutaneous lesions,lesions to tendons, lesions to ligaments, bone lesions, and lesions tomucous membranes.

According to another feature, the invention also relates to a method ofusing this pharmaceutical composition (i.e., suitable for localapplication, with or without antibiotic) as a medication for treatingfractures in mammals.

The present invention also includes a method of using the firstpharmaceutical composition described above (i.e., suitable forintravenous administration), as a medication for treating neurologicalor neurodegenerative pathologies in mammals including, withoutlimitation, Cushing's disease, head shaking, Wobbler's syndrome,breathing difficulties, and paresis of the limbs.

A further method of the invention relates to using this pharmaceuticalcomposition (i.e. suitable for intravenous administration), as amedication for treating acute or chronic inflammatory pathologies inmammals such as, but not limited to laminitis, periostitis, gastritis,arthrosis, and inflammations caused by viral, bacterial, parasite,and/or mycotic agents.

Finally, the invention also relates to a method of using the firstpharmaceutical composition described above (i.e., suitable forintravenous administration), as a medication for treating the syndromeof dilatation-torsion of the stomach in mammals. This pharmacologicalcomposition according to the invention may also be used for treatingpathologies of the gall bladder, cardiovascular pathologies, stress withconsequent depression and, in breeding, infertility of mares andprecocity of colts, and also for improving the competitive activities orperformances of mammals.

In each of the above methods, the expanded adult stem cells arepreferably contained in the pharmaceutical composition at aconcentration of about 150×10³ cells/ml and the medication isadministered once a week intravenously.

Preferably, the methods described above have particular application tothe veterinary field. The mammals treated may be horses, dogs, cats, orhumans, for example.

The cells isolated from peripheral blood act “in vivo” as pluripotentstem cells (PSC) following the expansion according to the invention andare able to resolve, within the space of a few months, lesions orpathologies incurable or curable only slowly with classicalmethodologies and/or drugs. The effects of the inventive method may beseen in conjunction with the following, non-limiting, examples.

Materials and Methods Sampling

Each sample of peripheral blood consisted of about 5-7 ml, was takenfrom the lower limbs of horses and dogs, and was immediately put intotest tubes containing, for example, heparin (150 U), and MCSF (10 nM).However, the heparin could be replaced by another suitableanti-coagulant substance.

Purification

The blood samples (5-7 ml) were diluted 1:5 in PBS (Phosphate BufferSaline) containing NH₄Cl (200 mM) to cause the lyses of the redcorpuscles, centrifuged at 10,000 g, washed twice with PBS, andcentrifuged again at 200 g. The nucleate cells obtained were incubatedfor 7-12 hours at 37° C., preferentially for 10-12 hours, and purifiedby means of fractioning on a Ficoll gradient, then isolated and washedthree times with RPMI medium 1640 (Life Technologies, Grand Island,N.Y.). Once purified, the cells that contained about 95% cells with CD90phenotype (as determined by means of cytofluorometric analysis by meansof a FACScan—Becton Dickinson flow photometer), were incubated foranother 24 hours in 50 ng/ml MCSF 45 nM, expanded to obtain the numberof cells necessary for the local or centrifuged treatments at 10,000 gand re-suspended in PBS at a concentration of about 90×10³ cells/ml forintravenous treatments.

Immunostaining

For cytophenotyping, the cells were washed in PBS and then fixed onslides in 4% formaldehyde in PBS for 20 mins at 20° C.

To identify the intra-cellular proteins, the cells were permeabilizedwith 0.5% Triton X-100 for 5 mins at 20° C. and then incubated for 1hour with the primary antibodies diluted in PBS containing 1% BSA (toblock the aspecific antigenic sites). After three successive washes, theslides were incubated for 45 mins with the secondary antibody conjugatewith the most appropriate fluorochrome: FITC or tetramethylrhodamine Bisothiocyanate (TRITC) or Cy5.

All of the secondary antibodies were developed, using the donkey ashost, by Jackson ImmunoResearch. The immunocytochemistries were carriedout at temperature of 4° C. and in a humidity saturated atmosphere.After three washes, the slides were mounted using “gelvatol-PBS”. Thefluorescence images were then acquired by means of a fluorescencemicroscope using as an internal standard an immunofluorescence directedagainst the glyceraldehyde 3-phosphate dehydrogenase (polyclonal sheepantibody produced by Cortex Biochem, San Leandro, Calif.). As negativecontrols and in order to calibrate the levels of the fluorescencebackground, slides incubated with aspecific antibodies of the sameisotype as the samples involved were used.

The images that were obtained show the cells seen through a phasecontrast microscope, superimposed on the fluorescence image of thelipids colored with Nile Red and on the image of the nuclei colored withDAPI (4′,6-diamidin-2-phenylindole). The reference bar measures 40 μm.The intensity of relative fluorescence was examined by means ofquantitative ratio imaging microscopy between cells treated with MCSFand macrophages.

The method described above was used to identify all the markersinvestigated (CD 90, CD 34, and CD90/CD34).

Results Local Use

The cells were applied directly in the cases of external wounds. On thecontrary, in the case of lesions to tendons, ligaments, and fractures,the cells were inoculated directly into the site of the lesion at afinal concentration, except where otherwise indicated, of 5-10×10⁶cells/ml according to the seriousness of the lesion. In cases where thecells could not be inserted precisely in the lesions the followingmethodology was followed. For lesions to collateral ligaments, theinjection was made between the second and third phalange. For lesions tothe naviculars, the injection was made in the carpal tunnel. Finally,for lesions to the sacroiliac articulations and in cases of Wobbler, theinjection was made between the fifth and the sixth cervical or betweenthe sixth and the seventh cervical.

Cutaneous Lesions

This involved the case of a mare with a wound between the metatarsus andfirst phalange, 20 cm in diameter with clostridia complications whichhad led to the destruction of the underlying tissues, including theextensor tendon (FIG. 1). The first application was made one year afterthe accident and after two surgical operations to remove the cheloids,and was repeated three more times at intervals of 15 days, using from 10to 400×10⁶ cells re-suspended in a physiological solution in thepresence of gentamicin. After 100 days, the wound was completely healed(FIG. 2), and after 6 months the hair had re-appeared in 70% of the scarzone (FIG. 3).

Tendons

Three horses were treated with lesions of 80% of the superficial flexortendon. After three months of treatment with about 300×10⁶ cells,hypoecogenic zones were no longer seen upon ultrasound exam. In fact, itwas observed that the thickness of the tendon (which had increased afterthe laceration and the inflammatory processes) was visibly reduced by80%, as can be seen from the ultrasound scans shown in FIGS. 4-7.

Other horses with smaller lesions to the tendon (1 cm in diameter) nolonger showed the lesion one month after treatment (FIGS. 8 and 9).

Ligaments

Among the lesions treated included an insertion of the suspenderligament under the hock with consequent lameness. Less than three monthsafter a local inoculation, the horse starting working again, withoutlameness, and after one year there was no relapse.

Other horses treated, which had lesions between the branches and thecentral body of the suspender ligament, front and rear, were allresolved with restitutio ad integrum.

Fractures

Among the fractures treated included the case of a dog with a fracturedfemur which, after 4 months following a surgical operation, was stillnot forming bone callus. After a local application of 10×10⁶ cells,there was a complete recovery in 30 days.

Mucous Membranes

With regard to the healing of lesions to the mucous membranes, variouschronic ulcers were treated. The most sensational case was that of adressage horse with several ulcers in the mouth which had already hadtwo plastic surgery operations. Following the treatment, the horsestopped bleeding within three days after local application of 4×10⁵cells. After 15 days, the lesions were completely healed.

These results clearly demonstrate that the local application of isolatedcells enriched by the inventive method on tendons, ligaments, joints andfractures provided complete resolution for more than 80% of the casesexamined within a few weeks or, at most, within four months. Theremaining 20% of cases showed considerable improvements. The methodscommonly used today in veterinary science in these cases give positiveresults in not more than 60% of cases after a treatment of about 6-15months, and improvements in only 5% of cases. Accordingly, the resultsachieved by the inventive methods are a dramatic improvement over priorart methods.

Intravenous Use

Cells were used intravenously (1 dose=150×10³ cells) in the followingpathologies:

Gushing's disease is a pathology due to hypertrophy of the intermediatehypophysis with reduced production of dopamine by the hypothalamus. Thiscondition is very similar to Parkinson's disease in humans.

An affected pony was treated, which also had, apart from the classicsymptoms of the disease, reduced immune defenses with hemolysis. Afterthree treatments at an interval of 5 days with 150×10³ cells pertreatment, improvements were noticed. After 4 cycles repeated atintervals of 40 days, the symptoms completely disappeared. The treatmentof another four horses in the same way produced the same results.

Head shaking is a neurological pathology of the central nervous systemwith secondary complications of the trigeminus, which leads tocontinuous head shaking and problems of photophobia. The horse treatedhad these symptoms for six months. The treatment entailed a cycle of150×10³ cells at intervals of one week for 5 weeks. Already in the thirdweek, the symptoms had disappeared. Two other horses treated with thesame protocol showed the same results.

Three cases of Wobbler. This is a congenital cervical neurologicalcompression. The cases treated by administering three doses(intravenously and local) at an interval of a week showed completeremission of the symptoms following treatment.

The intravenous use of expanded and purified stem cells using the methoddescribed showed how these cells are able “in vivo” to resolvepathologies affecting the neuronal tissue and to give results after aweek of treatment.

Vascular reconstruction. In a horse affected by laminitis (destructionof the peripheral vascularization in the foot, with the result of anextremely painful lameness such as to prevent movement), the pain hadalmost disappeared 12-24 hours after a dose was administered, inoculatedinto the digital vessels. The same result was obtained for two othercases of horses affected by the same pathology.

Other general cases in which the cells were administered intravenouslywere:

-   -   A case of a horse with a fracture of the navicular which        restarted its competitive activity after the simultaneous        administration of the stem cells in the peripheral vessels, the        navicular bursa and the articular surface between the deep        flexor tendon and the navicular bone;    -   A case of a horse suffering from periostitis which, with the        administration of two intravenous doses, improved its lameness;    -   A case of a seventeen year old horse operated for colic and        afterward put to grass because it was no longer able to continue        competitive activity due to a persistent state of general        debilitation. After four cycles of three doses every five days,        the horse started competitive activity again, participated in        competitions and obtained results it had never had before        achieved (FIGS. 10 and 11);    -   A case of a twenty-year-old horse with suspected cerebral        ischemia with consequent loss of coordination in three limbs        which, after pharmacological treatment based on cortisone        derivatives, still continued to keep an uncertain and staggering        pace. After the administration of two doses at an interval of        one week, the animal restarted its normal activity without        showing any symptoms;    -   A case of a twenty-three year old mare, with a pregnancy at 21        years of age, which, after the period of lactation had finished,        was treated with two doses of cells intravenously. After the        treatment the mare restarted her competitive activity in full.        (At 20, a horse is considered too old for competitive activity);    -   A case of a horse with a fractured pelvis which, after two doses        of cells, restarted international competitive activity;    -   A case of a fifteen-year-old horse with breathing difficulties        and a very nervy character, which, after treatment with two        doses, started competing again on an international level without        the slightest breathing difficulty;    -   A case of two horses with extreme lameness for more than two        years consequent to the pulling of the collateral ligament        between the first and second phalange. This pathology is        considered irreversible in 75% of cases. After treatment with        three doses for three months, both animals treated returned to        competing normally;    -   A case of a thirteen-year-old horse operated for colic at seven        and castrated at age twelve. Following the second operation, the        animal had non-emittent bacterial complications despite        treatment with antibiotics and anti-inflammatories. Moreover,        the animal suffered from chronic gastritis, proved by a        gastroscopy. After the administration of four doses at an        interval of one week, the horse was again given a gastroscopy        which showed the complete regeneration of the gastric mucous        membrane. The same result was obtained on ten other gallop        horses;    -   A case of a horse with a pansystolic murmur and numerous murmurs        atypical to cardiac osculation, with blood test positive to an        active form of Herpes virus 1 which caused neurological problems        and loss of balance. In this case, three intravenous doses were        administered three times at an interval of five days, followed        by a fourth dose administered at the level of the vertebral        column. Already after two months, the horse started to move        again, almost completely regaining balance;    -   A case of a nineteen-year-old horse almost completely retired        from competitive activity due to a general physical        deterioration, which, after the administration of 200×10³ cells,        twice at an interval of a week and with repeated treatment,        after three months returned to being one of the best in his        category (in jumping competitions, jumping heights of 1.35-1.40        m).

The same type of treatment was applied to very old dogs and gave thesame results. In dogs the following pathologies were treated:

Two cases of dogs with torsion of the stomach: the first was asix-year-old Great Dane subjected to a surgical operation but with arelapse after one week and therefore again subjected to anotheroperation. After a first administration of 150×10³ cells, the dogstarted eating again and, after two doses at an interval of one week,the dog returned to its normal activity.

The second case was a ten-year-old female Great Dane, suffering fromarthrosis, diagnosed with diabetes, and having had ahysterectomy-ovariectomy. Four months after the operation, she had atorsion of the stomach with a subsequent surgical operation which,however, did not bring a great improvement. At this point, two doseswere administered, at an interval of one week and four other cycles inthe following four months. Today, eight months after the last cycle, thedog not only has a normal glycemia but also her ability to walk hasimproved by 80%.

Finally, there was a case of a mongrel dog, 13 years old, male, withparesis of the rear limbs and incontinent. Until the treatment by themethod of the invention, it had only been treated with cortisone,without appreciable results. Fifteen days after the cortisone wassuspended, the cells were taken and the dog was subjected to a cycle oftwo doses at an interval of a week. Six days after the last treatment,the dog had not only reacquired the use of its legs, but was urinatingand defecating normally.

The results obtained by the method according to the invention make thisprocedure extremely versatile, thanks to the fact that, at the moment,no “in vivo” technique provides for expanding pluripotent cells.Moreover, the lack of any form of rejection or infection following theadministration in all the case histories reported above makes thistechnique suitable for auto-transplant procedures.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

1. A pharmaceutical composition comprising an active principlecomprising expanded adult stem cells and at least one of apharmacologically acceptable adjuvant and an excipient, wherein theexpanded adult stem cells are obtained using a method comprising: a)obtaining a sample of blood of an adult mammal; b) immediately after thesample of blood has been taken from the mammal, treating the sample ofblood in vitro with MCSF (macrophage colony stimulating factor) at aconcentration of about 8-15 nM MCSF to obtain expanded stem cells in thesample of blood; and c) purifying the expanded stem cells.
 2. Thepharmaceutical composition according to claim 1, wherein the expandedadult stem cells are contained at a concentration of about 90-250×10³cells/ml in the pharmaceutical composition, and the pharmaceuticalcomposition is formulated for intravenous injection.
 3. Thepharmaceutical composition according to claim 1, wherein the expandedadult stem cells are contained at a concentration of about 4-40×10⁶cells/ml in the pharmaceutical composition, and the pharmaceuticalcomposition is formulated for local or topical application.
 4. Thepharmaceutical composition according to claim 1, further comprising atleast one antibiotic as an active principle at a concentration of about5-15 nM.
 5. The pharmaceutical composition according to claim 4, wherethe antibiotic is gentamicin.
 6. The pharmaceutical compositionaccording to claim 4, where the antibiotic is amikacin.
 7. A method oftreating a lesion in a mammal, comprising administering to the mammal amedication comprising the pharmaceutical composition according to claim1, wherein the lesion is selected from the group consisting of acutaneous lesion, a lesion to a tendon, a lesion to a ligament, a lesionto a bone, and a lesion to mucous membranes.
 8. A method of treating afracture in a mammal, comprising administering to the mammal amedication comprising the pharmaceutical composition according toclaim
 1. 9. A method of treating a neurological or neurodegenerativepathology in a mammal, comprising administering to the mammal amedication comprising the pharmaceutical composition according to claim1, wherein the neurological or neurodegenerative pathology is selectedfrom the group consisting of Cushing's disease, head shaking, Wobbler'ssyndrome, breathing difficulties, and paresis of the limbs.
 10. A methodof treating an acute or chronic inflammatory pathology in a mammal,comprising administering to the mammal a medication comprising thepharmaceutical composition according to claim 1, wherein the acute orchronic inflammatory pathology is selected from the group consisting oflaminitis, periostitis, gastritis, arthrosis, and an inflammation causedby at least one of viral, bacterial, parasite, and mycotic agents.
 11. Amethod of treating a syndrome of dilatation-torsion of the stomach in amammal, comprising administering to the mammal a medication comprisingthe pharmaceutical composition according to claim
 1. 12. A method oftreating infertility in a mare, precocity in a colt, or for improvingcompetitive activity or performance in a mammal, the method comprisingadministering to the mare, the colt or the mammal, respectively, amedication comprising the pharmaceutical composition according toclaim
 1. 13. The method according to claim 7, wherein the expanded adultstem cells are contained in the pharmaceutical composition at aconcentration of about 150×10³ cells/ml, and wherein the medication isadministered once a week intravenously.
 14. The method according toclaim 7, wherein the mammals is selected from the group consisting of ahuman, a horse, a cat, and a dog.
 15. A method of preparing thepharmaceutical composition according to claim 1, comprising mixing theexpanded adult stem cells with the at least one of the pharmacologicallyacceptable adjuvant and the excipient.